: Human coronaviruses HCoV-229E and HCoV-0C43 cause up to 30 percent of upper respiratory disease in humans. HCoVs may also play a role in gastroenteritis and sinusitis, and coronavirus RNA has recently been detected in 44 percent of human brains tested. HCoV-229E infection is initiated by interaction of the viral spike (S) glycoprotein with its cellular receptor, human aminopeptidase N (hAPN), as discovered in Dr. Holmes' laboratory. The studies outlined in this proposal are designed to identify the receptor binding site of HCoV-229E S, characterize variant S proteins that can recognize non-human APN as a receptor, introduce specific mutations into the gene encoding the glycoprotein S. and analyze the resulting changes in receptor specificity. These studies wiH be accomplished by examining the genetic variation of S genes of HCoV-229E viruses passaged in cells expressing native APN glycoproteins of different species as well as chimeric and mutant APNs. Specific mutations identified in the variant S genes of HCoV-229E will be introduced into the gene encoding the viral S glycoprotein of a newly developed infectious cDNA clone of HCoV-229E. The resulting mutant viruses will then be evaluated for receptor specificity in human epithelial cell lines and non-human cell lines expressing recombinant and mutant hAPN.